Cross-feeding of bifidobacteria promotes intestinal homeostasis: a lifelong perspective on the host health

Throughout the life span of a host, bifidobacteria have shown superior colonization and glycan abilities. Complex glycans, such as human milk oligosaccharides and plant glycans, that reach the colon are directly internalized by the transport system of bifidobacteria, cleaved into simple structures by extracellular glycosyl hydrolase, and transported to cells for fermentation. The glycan utilization of bifidobacteria introduces cross-feeding activities between bifidobacterial strains and other microbiota, which are influenced by host nutrition and regulate gut homeostasis. This review discusses bifidobacterial glycan utilization strategies, focusing on the cross-feeding involved in bifidobacteria and its potential health benefits. Furthermore, the impact of cross-feeding on the gut trophic niche of bifidobacteria and host health is also highlighted. This review provides novel insights into the interactions between microbe-microbe and host-microbe.

Throughout the life span of a host, bifidobacteria have shown superior colonization and glycan abilities.Complex glycans, such as human milk oligosaccharides and plant glycans, that reach the colon are directly internalized by the transport system of bifidobacteria, cleaved into simple structures by extracellular glycosyl hydrolase, and transported to cells for fermentation.The glycan utilization of bifidobacteria introduces cross-feeding activities between bifidobacterial strains and other microbiota, which are influenced by host nutrition and regulate gut homeostasis.This review discusses bifidobacterial glycan utilization strategies, focusing on the cross-feeding involved in bifidobacteria and its potential health benefits.Furthermore, the impact of cross-feeding on the gut trophic niche of bifidobacteria and host health is also highlighted.This review provides novel insights into the interactions between microbe-microbe and host-microbe.
Bifidobacteria are abundant in the human gut and other warm-blooded animals, and are present in the rumen of ruminants, vagina 1 , intestines of honeybees 2 , oral cavity 3 , dairy products 4 , and breast milk 5 .Gut colonization by bifidobacteria occurs in the early stages of life and is closely associated with health and aging.Bifidobacteria are transmitted vertically from mother to offspring and are commonly found in the gut of healthy breastfed infants, accounting for 60-70% of all gut bacteria 6 .Aging alters the number and diversity of bifidobacteria in the human gut.In adulthood, the relative abundance of bifidobacteria decreases to approximately 10%, however remains stable, whereas that in aging individuals accounts for approximately 5% of the total gut bacteria 6 .In infancy, Bifidobacterium breve, B. longum, and B. bifidum are commonly found in feces 7 , while in adulthood, B. adolescentis, B. longum, and B. pseudocatenulatum are the dominant species 8,9 .The relative abundance of B. adolescentis decreases with age, whereas that of B. breve and B. longum is dominant in the gut of aging individuals 10 .
The rich and complex sources of carbon in the gut tract provide a trophic niche for gut microbiota.The differential utilization of glycans influences gut microbiota formation in gut niches, which may explain why certain species of bifidobacteria are more common in the gut tract of infants or adults 11,12 .Infant breast milk and adult diets contain a high abundance of glycans and oligosaccharides with complex structures that cannot be digested by the human body and hence pass through the large intestine as substrates for the gut microbiota.Bifidobacteria metabolize monosaccharides, disaccharides, and oligosaccharides; however, different bifidobacterial species prefer dietary glycans, including inulin-type fructan (ITF) 13,14 , resistant starch (RS) 15 , galactan 16,17 , xylan 18,19 , and arabinan 20 , while others utilize host-derived glycans, such as human milk oligosaccharides (HMOs) 21,22 and mucins 23 .
The metabolism of complex glycans by human gut microbiota is mediated by carbohydrate-active enzymes (CAZymes).Bifidobacteria has been estimated to use approximately 14.64% of the CAZyme gene, including glycosyl hydrolases (GHs) and glycosyl transferases (GTs), for the transport, degradation, and regulation of glycans, which is only slightly less than that of Bacteroides (20%).Gut microbiota employ similar strategies to maintain and break down complex glycans 24,25 .Bifidobacteria encode a series of modular glucanase complexes that are anchored to the cell surface by transmembrane domains.These contain genes encoding GHs to extracellularly digest long oligosaccharides or glycans and substrate-binding proteins (SBPs) of the adenosine triphosphate (ATP)-binding cassette (ABC) transport system to capture oligosaccharides digested before entering cells 26,27 .No strain has been identified to utilize all glycans; however, specific types of glycans are utilized.Metabolic specificity exists between different bifidobacteria and other gut microbiota in the range, species, and utilization strategies of available glycans, which induces interactions between bifidobacteria and other flora, including cross-feeding, to promote the gut adaptability of bifidobacteria 28 .
The utilization of HMOs by bifidobacteria is an example of occupying a specific trophic niche in the gut.B. longum and B. bifidum establish a crossfeeding relationship with other microbes (such as Eubacterium) in early life by sharing their HMO metabolites degraded by extracellular glycosidases, including fucose and short-chain fatty acids (SCFAs) 29,30 .This cross-feeding strategy can augment the accessibility of glycans to gut microbiota and mediate interspecific interactions through metabolite reuse, directly affecting host gut homeostasis and health 31,32 .
Understanding glycan utilization and cross-feeding mechanisms will help to facilitate the discovery of new genes, enzymes, and metabolites that are potentially involved in bifidobacteria-microbe associations and host health.Therefore, this review describes the glycan utilization properties of bifidobacteria and examines the glycan-based cross-feeding activities and mechanisms involved in bifidobacteria, as well as their impact on host health.

Glycan preference and metabolic pathway of bifidobacteria
The colonization advantages of bifidobacteria depend on the availability, demand, and consumption rates of specific nutrient resources.The different carbohydrate metabolisms of bifidobacteria in different periods influence their selective adaptation to different gut environments.Due to differences in trophic sources, the intestinal environment of infants differs from that of adults, characterized by bifidobacteria including B. longum subsp.infantis (B.infantis), B. breve, and B. bifidum, which use HMOs as the primary carbon source; however, when introducing complex diets after weaning, the bifidobacteria B. longum subsp.longum (B.longum), B. adolescentis, B. catenulatum, and B. pseudocatenulatum, which have a greater capacity to degrade plant glycans, are more prevalent 6,33,34 .The breakdown of glycans by bifidobacteria depends on a significant number of GHs located extracellularly or within the cell wall.Bifidobacteria have evolved several homologous enzymes with overlapping, yet different, substrate specificities.Different species have different intestinal adaptations to cope with different glycan structures.Complex glycans are first degraded by extracellular GHs into monosaccharides or oligosaccharides, some of which are transported directly to the cytoplasm by transport proteins, while others are phosphorylated.These monosaccharides or oligosaccharides, and other simple carbohydrates, enter the bifid-shunt pathway (fructose-6-phosphate phosphoketolase central fermentative pathway) of bifidobacteria and are metabolized and used for ATP production 35 .The metabolic pathways of bifidobacteria are closely associated with the type and concentration of extracellular sugars in the gut.Bifidobacteria normally produce various metabolites, including acetate, ethanol, and formate, depending on the amount of sugar available; when extracellular sugars are abundant, acetate and lactate are generated 36 .Therefore, bifidobacteria adopt different strategies to colonize, and the capture, degradation, and metabolism of glycans by bifidobacteria can determine their trophic niches in the gut tract (Fig. 1 and Table 1).

B. infantis primarily internalizes HMOs
The ability of B. infantis to intracellularly utilize most HMOs provides a competitive advantage over other HMO-consuming species (such as B. breve), resulting in its dominance in the infant gut during breastfeeding 37 .The processing of 2ʼ-fucosyl lactose (2ʼ-FL) by B. infantis depends on the ABC transporters for intracellular digestion.B. infantis contains several intracellular enzymes including fucosidase, β-galactosidase, LNB phosphorylase, N-acetyl-β-hexosaminidase, and sialidase, to degrade fucosylated and sialylated HMOs 38 .
Transporter specificity is necessary for the effective uptake of HMOs.Sakanaka et al. 26 have reported that the two FL transporters (FL1-BP and FL2-BP) of B. infantis can be used to absorb 2ʼ-FL and 3ʼ-FL, and the expression of FL transporters influences the abundance of B. infantis.Intracellular FL is utilized by α-fucosidase to produce fucose, which results in the secretion of 1,2-propanediol (1,2-PD), acetate, and lactate 39 .Nonfucose glycosylated neutral HMOs, including lactose-N-tetrasaccharide (LNT), lacto-N-neotetraose (LNnT), lactose-N-bisaccharide (LNB), and N-acetyl glucosamine, stimulate the growth of B. infantis 35,40 .Duar et al. 41 determined that the utilization of LNT and LNnT by B. infantis EVC001 is closely associated with the Blon2175-2177 ABC transporter in its H5 cluster, which contains a complete gene repertoire of HMO utilization, while the H5-negative strains exhibit growth defects on the carbon source.

B. longum can metabolize arabinoxylan
Colonization by B. longum dominates the entire lifespan of the host 42 .The abundance of B. longum is closely associated with food intake at different growth stages, while the gene pool for glycan acquisition and metabolism can be selectively altered due to changes in certain dietary components 43,44 .The colonization superiority of B. longum is manifested in its ability to grow on HMOs and metabolize complex arabinoxylan (AX) substrates.Some B. longum metabolize specific HMOs, including LNT and 2ʼ-FL rather than sialylated HMOs, as well as mucin O-glycans, thereby occupying trophic niches in the gut during early life 45 .Sakanaka et al. 45 identified an extracellular lacto-N-biosidase (LnbX) from B. longum JCM1217 that could consume LNT and release LNB and lactose via the GNB/LNB pathway.Díaz et al. 46 found that B. longum M12 with α-1-2-L-fucosidase (GH95) could grow on 2ʼ-FL and LNT.
The transporters and GHs of B. longum are more likely to metabolize plant-derived glycans (dominated by various arabinose-substituted glycans) 47 .The enrichment of the gene cluster encoding L-arabinofura (-Araf) facilitates B. longum utilization of these glycans and reflects its ecological adaptability and competitiveness in the adult and aging gut environments 48 .The GH43 family, which includes α-L-arabinofuranosidase, β-xylosidase, arabinosidase, and xylanase, is substrate-specific and degrades insoluble fibers through cooperation.B. longum has an abundant GH43 gene cluster capable of releasing arabinoxylooligosaccharides (AXOS), arabinose and xylose from arabinogalactan (AG), arabinogalactan (AN), and AX 16,49,50 .Collectively, the ability of B. longum to degrade AX and AXOS is strain-specific and influenced by different priorities for utilizing different monosaccharide compositions and structures (such as degree of polymerization (DP), linkage type, and sidechain space structure) 51 .For example, B. longum JCM 1217 may prefer natural and partially degraded AX components with higher DP, side chain content, and arabinosyl-monosubstituted structure, possibly due to differences in the transport system specificity and membrane localization of the enzymes 14 .AG and arabinogalactan protein (AGP) are complex fiber components in plant cell walls that are consumed only by a few sub-species of B. longum; most bifidobacterial species cannot absorb full AGP 52 .Fujita et al. 16 reported a type II AG-degrading enzyme in B. longum JCM1217 that can grow on larch AGP but cannot act on gum arabic AGP with a more complex structure.The high molecular weight arabinose side chain in the highly modified AGP causes steric hindrance, resulting in limited cleavage of β-1,3/1,6 bonds in the galactose skeleton by β-galactosidase and arabinoglycosidase and insufficient galactose and galactooligosaccharides (GOS) release 53 .Sasaki et al. 54 identified a novel arabinofuranosidase (named GAfase) that can cleave the l-Ara structure of gum arabic AGP to remove steric hindrance, a key factor in the growth of B. longum JCM7052 in gum arabic AGP.These relatively limited degradations suggest that B. longum must cooperate with other bacteria to completely consume the AGP and AG complex.Moreover, B. longum selectively utilizes other N-glycans for growth.Specifically, the α-mannosidases, N-acetyl glucosaminidase, and αglucosidase of B. longum NCC2705 release mannose and N-acetyl glucosamine (GlcNAc) from mannans through cooperation 55,56 .
B. breve has limited growth on complex HMOs HMO metabolism in B. breve is highly similar to B. infantis, which absorbs intact oligosaccharides through the ABC transport system and degrades them in cells.However, the complete gene pool required to metabolize complex HMOs, such as fucosyllactose and sialyl-lactose, and the utilization https://doi.org/10.1038/s41522-024-00524-6 Review article npj Biofilms and Microbiomes | (2024) 10:47 of HMO components by B. breve are limited to N-glycans, including LNT, LNnT, and N-disaccharide (LNB) 57,58 .The genome of B. breve mostly contains genes involved in the breakdown of lactose, fucose, sialic acid, and amino sulfate [59][60][61] .Specifically, the external sialidase (GH33) in B. breve can help to release sialic acid from mucin and grow on free sialic acid in the gut environment 29 .The N-acetyl glucosamine 6-phosphate deacetylase of B. breve UCC2003 can efficiently break down GlcNAc-6-sulfate residues in O-glycans 61 .Bottacini et al. 62 analyzed the genomes of 20 B. breve strains and determined that α-glucosidases (GH13 and GH31), β-glucosidase (GH1 and GH3), and β-galactosidases (GH2 and GH42) were involved in the metabolism of α-glucans (such as maltose), cellobiose, and galactose, respectively.Notably, most B. breve strains cannot utilize fucosylated HMOs (2ʼ-FL or 3ʼ-FL), but utilize fucose produced by other bacteria 63 .

B. bifidum extracellularly degrade HMOs and mucin O-sugars
Compared with other bifidobacterial species, B. bifidum has a complete set of extracellular GHs, including α-sialidase, α-fucosidase, N-Acetylβ-hexosaminidase, β-galactosidase, and LNBase, to assimilate complex HMOs and leave degradation products, such as lactose, fucose, and sialic acid, outside the cell 45,[64][65][66] .Nishiyama et al. 67 identified a sialidase (SiaBb2) involved in the degradation of HMOs and mucin and may promote the adhesion and colonization of B. bifidum on the surface of the intestinal mucosa.Additionally, B. bifidum lacks specific enzymes to degrade Nglycans, however, has a substrate preference for O-glycans, which avoids competition with other bifidobacterial species in the guts of breast-fed infants.B. bifidum encodes N-acetyl galactosidase (GH101) and Lacto-Nbiosidase (GH136), which degrade mucin O-glycosidic bonds, and has abundant carbohydrate-binding modules (CBMs) that promote the proximity of GHs to substrates 68,69 .Takada et al. 70 found that the β-Nacetylglucosaminidases of B. bifidum can specifically degrade β-GlcNAc linkages of mucin core structures, while Katoh et al. 71 reported that B. bifidum could release GlcNAc-6S from sulfated O-glycans using sulfoglycosidase and may affect the metabolism of other bacteria.
Similarly, the genome of B. catenulatum contains genes for GHs involved in HMOs and xylan, starch, and their derived oligosaccharides, which can adapt to changes in the host diet 86 .HMOs, including 2ʼ-FL and lacto-N-difucohexaose, are translocated intracellularly by B. catenulatum and degraded into GLcNAC and monosaccharides in cooperation with fucosidase, β-galactosidase, and Lacto-N-biosidases 87 .The extracellular xylanase of B. catenulatum can cleave AX into XOS and AXOS, which are subsequently catabolized by intracellular arabino-franosidase and xylosidase into arabinose and xylose 87 .

Glycan utilization capacity of bifidobacteria shapes cross-feeding interactions with other species
The gut microbiota is mostly auxotrophic, and the assimilation of glycans by different species may be similar (metabolic redundancy).Therefore, there remains a need to compete with other species or obtain nutrition from the gut environment 88,89 .Relying on other microorganisms that release metabolites into the environment, competing species can coexist in equilibrium; these cost-free metabolites promote microbial interactions 90 .Cross-feeding exists between species with different metabolisms, specifically those that utilize particular complex compounds to liberate metabolic byproducts that are further assimilated by others that cannot grow on those compounds alone 28,90,91 .Bifidobacteria rely on their enzymatic breakdown and oligosaccharide transport systems to consume glycans from the host, resulting in stable colonization of the gut tract and facilitating cross-feeding relationships within bifidobacterial species or with other bacteria, such as Bacteroides and butyrate producers 92 (Fig. 2  and metabolites (such as 1, 2-PD and acetate) that play important roles in the gut tract of breastfed infants by cross-feeding with other bacteria such as Eubacterium hallii and Anaerostipes caccae 30,93,94 .Cheng et al.95 found that B. infantis utilizes 6ʼ-sialyllactose (6ʼ-SL) for acetate production and is cross-fed to Faecalibacterium prausnitzii for butyrate conversion, which causes B. infantis to proliferate as a result.This may be associated with the fact that F. prausnitzii secretes extracellular sialidase to promote the expression of sialidase in B. infantis, thereby increasing acetic acid production, and that the occurrence of this crossfeeding activity depends on the molecular structure of HMOs 95,96 .
Cooperative degradation of HMO between bifidobacterial species.
When extracellularly degrading HMOs, the products produced by bifidobacteria (including B. bifidum and B. longum) are partially released into the public environment, inducing cross-feeding with other bifidobacterial species with weaker HMO-utilizing capacity.
For example, the products of LNnT degradation by B. longum could be cross-fed to B. pseudocatenulatum, and the oligosaccharides from 2ʼ-FL degradation by B. pseudocatenulatum support the growth of B. longum 29 .B. bifidum is not the dominant species in the intestinal tract of infants; however, as an extracellular degrader of HMOs, it cross-feeds with other species by releasing HMO derivatives, thus affecting the composition of the gut microbiota 97 .HMO metabolism between B. bifidum and B. breve is complementary, which also contributes to cross-feeding between the two strains 61 .B. bifidum uses fucosidase and FL transporters to release lactose and fucose extracellularly, which cross-feeds to B. breve.This indicates that B. bifidum is easy to cross-feed with other fucose consumers who lack extracellular fucosidase.However, B. bifidum prefers to grow on lactose rather than fucose, suggesting altruism as its primary function 98 .In addition to cooperation, B. breve can compete with B. bifidum for lactose; the key to coexistence is that B. bifidum releases more lactose by upregulating the gene expression of the enzymes and transporters involved 98 .A similar phenomenon was observed in the co-culture of mucin and sialylated glycan 59,99 .Chen et al. 100 demonstrated that B. bifidum released sialic acid and lactose from sialylated glycans, which supported the growth of B. infantis and B. breve as secondary metabolites of cross-feeding.B. bifidum does not assimilate fucose, galactose, NeuAc, and GlcNAc-6S; therefore, the degradation of mucin O-glycan may be accomplished in cooperation with other species, such as B. breve, that utilizes these mucin-derived carbohydrate fragments 66,101 .HMO-based cross-feeding of Bifidobacteria with other gut bacteria.
Cross-feeding relationships established between bifidobacteria and other gut bacteria can help to refine the network of interactions within the gut microbiota during early life.Cheng et al. 102 demonstrated that the metabolite 1,2-PD produced by B. breve through fermenting fucose could promote the colonization of Limosilactobacillus reuteri in the gut of gnotobiotic mice.Nogacka et al. 103 investigated the 2-ʼFL-based metabolic interactions between B. bifidum and Lactobacillus gasseri and found that B. bifidum IPLA20048 promoted the proliferation of L. gasseri IPLA20136 by cross-feeding the extracellular degradation products (galactose, fucose, and lactose) and that genes encoding α-fucosidase involved in carbohydrate transport are upregulated in B. bifidum thereby increasing carbohydrate production.
The gut microbiota of breastfed infants undergoes significant changes in response to diet before and after weaning, thereby facilitating more complex microbial interactions and inducing gradual stabilization and maturation of the structure and composition of the gut microbiota.Phytoglycan-based cross-feeding of bifidobacteria after weaning enables its persistence in the gut Cross-feeding of bifidobacteria with butyrate producers based on plant glycans.Acetic and lactic acids are the end products of oligosaccharides metabolism by bifidobacteria and are important substrates of cross-feeding between bifidobacteria and butyric acid bacteria [104][105][106] .When grown on AXOS, except for the conversion of acetic acid to butyric acid, butyrate producers consume AXOS to produce arabinose and xylose, which can be consumed as substrates by B. longum 107,108 .Bhattacharya et al. 109 investigated the cross-feeding between B. adolescentis and Roseburia based on galactose-substituted β-mannanoligosaccharides.
Moens et al. 110 confirmed that bifidobacteria can easily cross-feed on FOS and inulin-type fructan to produce the bifidogenic and butyrogenic effects and that the conversion degree of acetic acid to butyric acid was closely associated with the differences in the glycan-degrading ability of bifidobacterial species.Specifically, when co-cultured with F. prausnitzii, B. breve can only utilize fructose, cannot consume FOS and ITF, and relies on the monosaccharides produced by F. prausnitzii to degrade fructan for growth.B. adolescentis prefers short-chain FOS over ITF, allowing it to cross-feed acetate to F. prausnitzii and utilize its FOS for continued growth.In contrast, B. longum or B. angulatum competes with F. prausnitzii on ITF, resulting in the lowest efficiency of butyric acid synthesis in the co-culture system 110 .Phytoglycan-based cross-feeding between bifidobacterial species expands carbon source availability.When co-cultured, bifidobacterial species that cannot degrade plant glycans rely on other species that can degrade.For example, Centanni et al. 111 reported that when grown on Hi-Maize, B. pseudolongum extracellularly produced type 1 pullulanase and alpha-amylase for the release of glucose, maltose, and maltotriose, which are cross-fed to B. animalis.More importantly, bifidobacterial species with similar nutrient metabolism can expand their trophic niches by cross-feeding the same substrates rather than competing.For example, B. bifidum PRL2010 lacks a degradation system that utilizes plant glycans, such as starch and xylan; however, when co-cultured with B. adolescentis 22 L, B. breve 12 L, and B. thermophilum JCM1207, it can grow on simple carbohydrates released by other bifidobacteria and promote its sugar utilization gene expression 112 .When co-cultivated with other bifidobacteria, B. adolescentis exhibits the most significant alterations in genes involved in xylose metabolism compared with other species, reflecting the enhanced genetic adaptability of these strains, which may help hosts to expand the utilization potential of carbohydrates during specific dietary changes 113 .
Bifidobacteria cooperates with other gut bacteria to degrade complex plant glycans.Cross-feeding of breakdown products of complex glycans among the gut microbiota depends on the complexity of the target glycan.Due to the lack of a complete degradation and transport system, bifidobacteria have a limited capacity for recalcitrant plant glycans, including xylan, long-chain inulin, and mannan; therefore, they rely on other bacteria to extracellularly release soluble oligosaccharides as cross-feeding substrates, such as AXOS, XOS, FOS, and mannose 76,114 .Some members of the genera Bacteroides and Lactobacillus are dominant glycan-degrading bacteria that can cross-feed with Bifidobacterium, including B. longum, B. animalis, and B. adolescentis 19,115 .When grown on simple xylans, such as glucuronoxylan and wheat arabinoxylan, B. adolescentis ATCC 15703 relies on AXOS produced by Bacteroides ovatus ATCC 8483 for growth in the absence of a degradation system containing xylanase (GH98) 116 .The cross-feeding of fructans is closely associated with the substituents and length of the sugar chain, and complex fructans (i.e., those containing sucrose units) are degraded by primary degraders to produce short-chain compounds that are utilized by secondary consumers 117 .B. longum tends to use short-chain inulin, leading to cooperation with Lactobacillus paracasei to completely degrade longchain inulin and produce fructose, lactate, and acetate 118 .B. longum has a cross-feeding relationship with Bacteroides based on xlyan, type II AG, and AGP.Wang et al. 53 reported that when co-cultured with Bacteroides caccae ATCC 43185, B. longum NCC 2705 thrived on the carbohydrate fragments of larch wood AG degraded by B. caccae.Vega-Sagardía et al. 119 reported that B. ovatus HM222 degraded xylan and promoted the growth of B. longum PT4 by releasing XOS and extracellular enzymes and that the secretion of α-L-arabinoglycosidase, L-arabinose isomerase, and xylulose isomerase increased in B. longum PT4.However, due to the poor growth of Bacteroides at pH <5.5, with the production of acetate and lactate by B. longum fermentation, the substrate conversion (from succinic acid to propionate) efficiency of Bacteroides decreases, resulting in succinic acid accumulation 53 .Therefore, cross-feeding with Bacteroides is pH-limited, and the combined bacteria used to degrade complex AG or xylan require the participation of other species, such as Prevotella and Roseburia intestinalis 120,121 .Notably, the cross-feeding relationship between B. longum and Bacteroides is mutual, and the secondary products of plant glycan metabolism from B. longum can be cross-fed to some Bacteroides with a poor glycan metabolic system.For example, AGPrelated oligosaccharides degraded by B. longum can be cross-fed with Bacteroides vulgatus, which promotes xylosidase expression in the latter strain 122 .Sonnenburg et al. 123 determined that B. longum promoted the functions of mannosidase and xylosidase in Bacteroides thetaiotaomicron when co-cultivated on plant glycans.Given the limited data, whether the cross-feeding interactions involving bifidobacteria contribute positively to the balance of carbohydrate utilization within the entire intestinal community remains to be determined.
Availability of glycans and cross-feeding activities shape the critical position of Bifidobacterium during life span The cross-feeding activities produced by bifidobacteria underscore their key ecological role in acquiring or providing substrates from or for other bacteria.This dependent interaction can also enhance the metabolic function of the participants, thus expanding their original trophic niche.The supplementation with bifidobacteria-containing multistrain synbiotics to construct a network of interactions between different members of the gut can help the gut microbiota to mature and stabilize during early life and increase the secretion of beneficial metabolites (such as butyrate), ultimately preventing chronic diseases associated with an imbalanced gut microbiota 44,124 .This synergistic combination of strains synergistically regulates gut microbiota and health homeostasis and is less affected by endogenous microorganisms than a single strain 125,126 .However, current research on the regulation of health by multi-strain synbiotics is only at the phenomenal description level, and clear explanations of the underlying reasons for the combination, the basis of its formulation, and the mechanism of its effectiveness remain scarce.Therefore, the possibility and necessity of such multi-strain synbiotics should be explored based on the preference of probiotics for glycans and the cross-feeding their relationships 127,128 .Gut microbiota is often affected by age, dietary habits, and antibiotics, necessitating tailored nutrition based on the needs of different hosts using appropriate and individualized strategies to regulate flora homeostasis 34,129 (Fig. 3).Beneficial effects of cross-feeding involved in bifidobacteria during early life Ameliorating the adverse consequences of microbial colonization caused by cesarean section and antibiotic use.Mode of birth (cesarean section) and antibiotic treatment negatively impact gut microbiota during infancy, as evidenced by the accumulation of pathogenic bacteria, decrease in Bifidobacterium and Bacteroides, and increase the risk of metabolic, inflammatory, and immune disorders in infants 127,130,131 .Supplementation with Bifidobacterium strains with extensive HMO metabolism (such as B. infantis), probiotics, and prebiotics that can stimulate the growth of Bifidobacterium have been used to reverse gut microbial dysbiosis induced by childbirth 132,133 .B. breve has been reported to grow well on 2ʼ-FL by coexisting with other species encoding an extracellular fucosidase (GH95).Lou et al. 132 found that supplementation with R. gnavus can promote the proliferation of B. breve, which utilizes lactose released by the former from the 2ʼ-FL, and contribute to the shift of the preterm microbiome to a bifidobacteria-rich community.Korpela et al. 127 developed a mixture of Lactobacillaceae rhamnoides, B. breve, and Propionibacterium freundenreichii combined with FOS, which promoted the colonization of Bifidobacterium and reduced the abundance of Enterococcaceae, Clostridiaceae, and Veillonellaceae, ultimately correcting adverse changes in the gut microbiome due to cesarean section and antibiotic induction.A metaproteome analysis revealed that the enzymes used for HMO degradation in Bifidobacterium (including β-galactosidase and β-galactosyl N-acetyl hexosaminephosphorylase) were significantly expressed and Lactobacillus rhamnosus and might benefit from the monosaccharides released from HMO degradation 127 .These studies suggest that the administration of probiotics and/or prebiotics with gutresident species (especially Bifidobacterium) as allies is effective in restoring gut microbiota disorders in infants; such multi-strain and prebiotic combinations with synergistic effects can be used as supplements in infant formulas.
Promoting a dominant position of bifidobacteria and perfecting intestinal community assembly.HMO-based metabolic interactions can shape the composition of infant gut microbiota.Bifidobacterium (especially B. infantis) is prioritized for gut colonization of breastfed infants, as determined by their superior ability to assimilate HMOs 21,134 .Bifidobacteria exert a priority effect based on HMO metabolism shortly after birth and consume most of the carbon sources in the gut, which prevents the colonization of later species, stimulates the establishment of infant intestinal homeostasis, and inhibits the growth of pathogens (such as Clostridium difficile and Salmonella) 135 .Intestinal colonization of B. breve is an example of priority effects.Considering the limited utilization ability of B. breve toward HMO, the formation of its dominant position is not simply related to the phenotype of HMO consumption but depends on cross-feeding with other bifidobacteria 61,66 .
The cross-feeding activity involved in bifidobacteria plays a key role in the assembly of gut microbiota.Chang et al. 136 found that B. infantis Bg2D9 promoted the colonization of Prevotella copri Bg131 in the gut of malnourished mice and facilitated the release of arabinose from diets containing arabinoglycans, which contributed to the proliferation of other species in the gut, including B. catenulatum and Blautia obeum, by cross-feeding.This could be used as a dietary intervention in severely acutely malnourished children.
In summary, the gut microbiota is highly malleable after birth and it is possible to promote gut homeostasis by introducing diets with appropriate synbiotics.Determining the processes and outcomes of these interactions can be used for dietary interventions to achieve personalized nutrition 137 .

Beneficial effects of cross-feeding involved in bifidobacteria during adulthood
Reestablishing gut microbiota structure and gut homeostasis.The prevalence of endogenous bifidobacteria is lower in adulthood than early life; however, due to the increase in dietary diversity, cross-feeding activities involved in bifidobacteria facilitate more interactions with the gut microbiota and host 34 .Dietary supplementation with multi-strain synbiotics helps to promote the proliferation of bifidobacteria and to reestablish a stable gut community 138,139 .Exposure to antibiotics can disrupt the abundance and diversity of the gut microbiota, and the introduction of deleted core microbiota can help to rebuild the gut immune homeostasis through syntrophic relationships 125 .For example, Button et al. 140 developed a synbiotic of HMOs and B. infantis to improve the imbalance in the gut microbiota and affect the growth of Enterobacteriaceae by promoting the production of acetate and butyrate.A follow-up study by Button et al. 141 found that this HMO-dependent bifidobacterial strategy could increase the abundance of lactate consumers (Veillonella spp.) and alleviate antibiotic-induced dysbiosis of the gut microbiota by decreasing the pro-inflammatory metabolite p-cresol sulfate.
Stimulating secretion of beneficial metabolites and improving metabolic status.Due to the cross-feeding relationships with butyrate producers, changes in the abundance of Bifidobacterium are strongly correlated with fecal acetate and butyrate concentrations, and their metabolic interactions can modulate metabolic syndromes such as type 2 diabetes (T2D) and obesity 142,143 .Butyrate plays an important role in the regulation of blood glucose levels 144 .Perraudeau et al. 145 designed a synbiotic containing Akkermansia muciniphila, B. infantis, three butyrate producers, and inulin to improve T2D and determined that the cross-feeding activities of A. muciniphila and B. infantis with butyrate producers promoted butyrate production, which warrants in vivo validation.Multistrain synbiotics have also been used to improve energy metabolism, immune function, and gut microbiota in individuals with obesity 146,147 .Kanazawa et al. 142 introduced a synbiotic composed of GOS, B. longum, and B. bifidum to regulate obesity, and determined that an increase in Ruminococcus can be used for butyrate production.Nguyen et al. 148 used arabinoxylan to treat obesity through co-occurrence network analysis and found that B. longum released oligosaccharides through the degradation of arabinoxylan for synergistic and mutual interactions with Bacteroides, Phascolarctobacterium, and Subdoligranulum to promote the production of acetate and mitigate obesity.Dietary changes and synbiotic interventions can be used to increase the abundance of bifidobacteria in the gut tract and promote the secretion of beneficial metabolites, thereby mitigating metabolic disorders; however, the clinical significance remains to be determined.Future perspectives.Nutrition changes, such as those from breast milk in early life to complex glycans in adulthood and beyond, directly determine the evolution of bifidobacteria in the gut.The glycan preference of bifidobacteria is useful to adapt to changes at different ages and dietary stages and introduce cross-feeding relationships between bifidobacterial strains and other gut microbiota, providing positive feedback to the host.Therefore, the glycan utilization strategies of Bifidobacterium and their cross-feeding networks must be explored.However, unlike Bacteroides, evidence of the intracellular and extracellular GHs and transport systems of Bifidobacterium remains scarce.
The ability of the transport system to recognize specific substrates may influence cross-feeding, which warrants further investigation.In addition, cross-feeding between Bacteroides and Bifidobacterium based on AX and AG has been reported; however, the evidence remains inadequate, and the syntrophic relationship with other glycans, such as β-glucans and fructans, remains to be elucidated.Future studies should further investigate the crossfeeding of bifidobacterial species (such as B. longum and B. pseudocatenulatum) and that between bifidobacteria and gut microbiota (such as Lactobacillus).
The prevalence of bifidobacteria decreases with age.The cross-feeding activities of bifidobacteria are beneficial to the host; therefore, dietary intervention strategies must be adopted to regulate health status.However, most existing studies on the health benefits of multistrain synbiotics have focused on the outcome of the intervention rather than the process and lack the scientific basis for such synergistic combinations.This necessitates the development of individual formulas and consideration of intervention times based on the gut microbiota of individuals at different ages or nutritional stages.The beneficial effects of bifidobacteria involved in multistrain synbiotics and strategies to alter the relationship between gut microbiota and host must be determined.The targeted modulation of bifidobacteria through cross-feeding strategies can shift the classic nutritional studies of flora to molecular nutrition, which will be the future trend of microbiome therapy.

Fig. 1 |
Fig. 1 | Major glycan utilization strategies of bifidobacteria.Bifidobacteria complete the degradation by cleaving the glycan by extracellular enzymes or internalizing the glycan directly.B. infantis can internalize most of the HMOs through the transporter system, and B. bifidum degrade the HMOs (including fucosylated glycans) by abundant extracellular enzymes for further internalization and uptake.B. breve utilize limited HMOs (mainly 2ʼ-FL).B. longum excel in the utilization of phytoglycans containing arabinose structures.B. adolescentis is an excellent utilizer of resistant starch.Both B. pseudocatenulatum and B. catenulatum can consume some HMOs and phytoglycans.

Fig. 2 |
Fig.2| Cross-feeding strategy of bifidobacteria throughout the life span.Crossfeeding of bifidobacteria exists throughout the life cycle.The major categories are HMO-based and phytoglycan-based cross-feeding.Specifically, when co-cultured on HMOs, bifidobacteria are more likely to cross-feed HMO degradation products to other non-HMO degradation-dominant bacteria and butyrate producers.When cross-fed on phytoglycans, bifidobacteria are more likely to thrive by relying on the extracellular degradation products of other dominant degrading bacteria.

Fig. 3 |
Fig. 3 | Benefits of cross-feeding of bifidobacteria throughout life span.The bifidobacteria of the host are influenced by several factors from birth.Breastfeeding and the use of probiotics and prebiotics contribute to the establishment of a network of interactions of the gut microbiota, including cross-feeding.The cross-feeding relationship can facilitate the formulation of multi-strain synbiotics, which are effective in improving intestinal homeostasis and mitigating related disorders including dysbiosis, metabolic syndrome, and immunodeficiency, due to cesarean section and antibiotic use.
6,80,79d that B. adolescentis metabolizes glucuronosylated-XOS (GXOs) and XOs (DP 2-6) to produce SCFAs.B. adolescentis prefers (A)XOS to arabinose or xylose, and encodes a gene for endo-1,4-β-xylanase capable of growing on wheat AX9,14,79.B. pseudocatenulatum and B. catenulatum consume host and plant glycans B. pseudocatenulatum and B. catenulatum are found in the guts of infants and adults.Their CAZyme genes differ in hosts of different ages6,80, suggesting that both species have a relatively well-developed degradation system for both host and plant sugars.
and Table2).HMO-based cross-feeding of bifidobacteria allows for its dominance in the guts of infants Cross-feeding of bifidobacteria with butyrate producers based on HMOs.B. infantis metabolizes HMOs to produce oligosaccharides Table 1 (continued) | Major glycan utilization strategies of bifidobacteria npj Biofilms and Microbiomes | (2024) 10:47